Interfleukin?7(lIL-?7) has been demonstrated to provide signals via the IL-?7 receptor which are requiredatmanysttagesofllymphocyttedevelolopment.t. IL-7 receptor is composed of two different cytokine receptor subunits;the IL-?7 receptor alpha chaininandthecommongammachainin.. These two subunits are shared with other cytokine recepttorrs:: IL-7? receptor alpha chain is also asubunit of the functional thymic stromal lymphopoiesis receptor, while common gammachainisisa component of functional receptor complexesfor IL-2,,IlL-4,, IL-?7,, IL-9,, IL-15,,and IL-21.. The developmental processes governed by IL-7? receptor signaling include initiation offcell proliferation, protection from apoptotic cell death, and the induction oflineage-speciifiicevents, such as gene rearangement in antiigen receptor loacdi. We have previously shown that cell survival signaling mediated by IL-7-driven upregulation of an anti-apoptotic protein, Bcl-2, is important for I cell development. On the other hand, IL-7 plays a critical role in regulation of EBF expression at an early phase of B cell development in adult bone marrow. Dysfunction of IL-7 receptor subunits (either 11-7 receptor alpha or common gamma chains) causes profound immunodeficiency in both humans and mice, highlighting the central role IL-7 plays in lymphopoiesis. Although progress has been made in identifying the intracellular mediators of 11-7 The specific aims for the 2 year term are as follows: a.a. region of Il-? receptor alpha in B cell development;and 3) to clarify the role of Il-?R and Flt3 in EBF expression during B cell development. The goal of this ARRA project is to elucidate the signaling pathways downstream of IL-? receptor, which drive specific steps in lymphocyte development, and to determine the genes involved in this process. moo 1..- c=-' -_' o05 c[unreadable]- -[unreadable]r-0 f-" expression at an early phase of B cell development in adult bone marrow. Dysfunction of IL-? B cell potential in pre-proB/CLP2 cells;2) to determine importance of 414-441 important for T cell development. survival Signaling mediated by IL-?-clriven upregulation of an anti-apoptotic protein, Bcl-2, is immunodeficiency in both humans and mice, highlighting the central role IL-? plays in receptor subunits (either IL-7 receptor alpha or common gamma chains) causes profound receptor signaling that regulate these developmental processes. significant gaps remain. lymphopoiesis. Although progress has been made in identifying the intracellular mediators of Il-7 On the other hand. IL-7 plays a critical role in regulation of EBF 1) to determine the role of IL-? in maintenance of We have previously shown that cell chi -to >_L.+ ;'0 vii A'0 .V3 receptor signaling that regulate these developmental processes, significant gaps remain. The and to determine the genes involved in this process. pathways downstream of IL-7 receptor, which drive specific steps in lymphocyte development, expression during B cell development. The goal of this ARRA project is to elucidate the signaling receptor alpha in B cell development;and 3) to clarify the role of IL-7R and F1t3 in EBF B cell potential in pre-proB/CLP2 cells;2)to determine importance of 414-441 a.a. region of 11-7 specific aims for the 2 year term are as follows: 1) to determine the role of IL-7 in maintenance of E ='3 QED v-- -to :;:- 0)0 Modified Specific Alms Section A. Specific Aims Interleukin ?7 (IL-?7) provides signals, via the 1IL1--?7recepttorr(IlL-7R)),,whiichare required during many stagesof lymphocytedevellopment.. IL-7R is composed of two different cytokine receptor subuniitts,,IlL-?7Ra. and commony((Y,c') chains.. IIL-7?Raiisalso acomponent of the thymic stromal Ilymphopoietin (TSLP) receptorcompllex,. whereasYc iscommon to functional receptor complexes for IL-2, IL-4., lIL-7?, 1IL1-9.,IL--1S5.,and I1L1-2-211. . The developmentall processes governed by lIL-7R signaling include the initiation of cell proliferation, protection from apoptotic cell death,. and the inductiionofliineage-speciifiiceventts(ii..e.,generearrrangementtininantitgigenrreceptotorrloloci)i.). Mutations that interfere with IL-7R signaling cause profound immunodeficiency in both humansandmiice,. highliighttiningtthecenttrrallroleleIlL-?7pllaysinllymphopoieiesisis.. We recently found that StatS5-mediated EBF expression in pre-proB cells in response to IL-7 is indispensable for the stage transition from pre-proB to proBcellsls.. As aconsequence of IlL-?7 stimulatiion.,Statt5becomesacttivivateted.. Since the constitutively active form of StatS5 can rescue impaiiredBcellldevellopmentiinttheabsenceoff IL-7R.,SttatSt5pllaysacenttrall rolleiinIlL-7R signalliningiinBcel!lldevelolopment.t. EBF forms a transcription factor network, which is necessary for B cell specification and commitment along with other B cell transcription factors, such as E2A, P11.1, and Pax5. Therefore, IL-7 is an indispensable extracellular factor that regulates B cell specification prior to the B lineage commitment stage. On the other hand, IL-7Rc('fetal liver pre-proB cells express comparable levels of EBF to wild type (WT) pre-proB cells in adult bone marrow. B cell development in EBF4 fetal liver is blocked at the pre-proB stage as is the case with adult IL-T'or lL-7Rc mice, suggesting that factors other than 11-7 can upregulate and maintain EBF expression in fetal liver. We hypothesize that F1t3 ligand can substitute for IL-7 function in fetal liver since F1t3 can also activate Stat5. Although Stat5 is a central player in l1-7R signaling, it seems that other signaling molecules via 1L-7R play an important role in fymphopoiesis. We found that an IL-7Ra mutant that lacks the 414-441 a.a. region cannot support B cell development despite Stat5 activation. Therefore, it is critical to identify the molecules that associate with the 414-441 a.a. region of IL- 7Ra. In addition, lL-7 stimulation is necessary for B cell progenitors transitioning from the CLP to -ti 1,o00Q nom' ton am= c?m (AA '[unreadable]', 177 coo 6-0 'N+ Therefore, it is critical to identify the molecules that associate with the 414-441 In addition. Il-7 stimulation is necessary for Bcell progenitors transitioning from the CLP to a.a. region cannot support B cell development despite 8tat5 activation. [unreadable]O. that lacks the 414-441 7Ra. We hypothesize that Flt31igand can substitute for IL-? function in fetal liver since Flt3 can also suggesting that factors other than IL-7 can upregulate and maintain EBF expression in fetal liver. fetal liver is blocked at the pre-proB stage as is the case with adult Il-T'- or IL-?Ra-l- mice, levels of EBF to wild type (Wl") pre-proB cells in adult bone marrow. molecules via IL-7R play an important role in lymphopoiesis. commitment stage. activate Stat5. with other B cell transcription factors. such as E2A, transcription factor network. which is necessary for B cell specification and commitment along indispensable extracellular factor that regulates B cell specification prior to the 8 lineage min Although Stat5 is a central player in IL-7R signaling, it seems that other signaling On 'the other hand, IL-?Ra-l- fetal liver pre-proB cells express comparable PU.1, and Pax5. We found that an IL-?Ra. mutant B cell development in EBF-I- Therefore, IL-7 is an a.a. region of IL- ((]p _Mw C:@ Sit Mac 30)'a=' m0@ "mom >t1 Wpm N=m cum !17 -i. }+. pound0O0OIlIjI S1DV1P4OD 5 S1NVIO SLZ9 1tS89 6166XVXdVdLpoundL:pound:1 IDHidd6060OZOI/S1l/SO0 (OO:~hO-l1W~9) kWJd9g~li:L~. 60O0Ol3//~~L/~O well as overall general lymphoid potential in pre-proB cells.. The Specific Aims are as follows: 7R function mainly mediated by Stat5, which may synergize with Flt3 in B lineage specification as dendritic cell differentiation potential, we will examine whether these potential are maintained in IL-7-!- pre-proS cells. pre-proB stage in order to maintain B cell potentiiall.. Since pre-proB cells also have T and 7R function mainly mediated by Stat5, which may synergize with F1t3 in B lineage specification as lL-7" pre-proB cells. Therefore, the goal of this ARRA ROl project for 2 years is to elucidate IL- dendritic cell differentiation potential, we will examine whether these potential are maintained in Therefore, the goal of this ARRA R01 Since pre-proB cells also have T and project for 2 years is to elucidate IL- The Specific Aims are as follows: E-' om . a F"" >.m ?-- l(D Aim 1. TodetetermrmininettherroleleofflLIL--7ininthemaintenance of lymphoid developmental potential in pre-proB/CLP2 cells. a) Determination of TI celll,Bcell, and dendritic cell potential in IlL-T"'prere--prroBcellls. 9b) T&o o9xas"m,iRinSe tAhSe e8ftffe8c~t osfthRe-cGo8An~sititwutiv..e.asaotilv.'9o fo9r"m.. osf pound;taotl5i-aQnRd~ N~JoetoahR inA B8 aSnAda Ts8oaIl p&o~tseRlnitaial,l,Froecspseaotti\ifvselyt,y,iAinII.I.L77RII&!.~or IL T'ppror9ppror:BeBp8o9l1I1oe. Aim 2. Todetetermrminineththeimimportratanncceeoffththe414-441a.a. region of IlL.-7Rau in B cel development. a) Examination of Stat5 actMty in cells with WT and the 414-441 mutant after IlL-7 stimulation. b) Cc) d) a.a. region by pull-down and mass spectrometry assays. Examination of Stat5 protease activity in response to IL-7. Identification of association molecules to the 414-441 a.a. region by pull-down and mass Investigation of the role of the proteolysis resistant form of Stat5. ti) spectrometry assays. Investigation of the role of the proteolysis resistant form of Stat5. Examination ofStat5 protease activity in response to IL.7. Identification of association molecules to the 414-441 Examination of Stat5 activity in cells with WT and the 414-441 stimulation. ''~p Aim 3. development. Aim 3. To clarify the role of IL-7R and Flt3 signaling in EBF expression during B cell development c) a) c) b) a) s) Analysis of EBF expression levels in B cell progenitors derived from Flt3L..,t.IL_T'. mice. i!xliI",iAfiti8A sf'R8 ,ffe&t sf SA asti..., To clarify the role of IL.7R and FIt3 signaling in EBF expression during B cell -l. mice. .t.. Analysis of EBF expression levels in B cell progenitors derived from FIt3L'iL-T'mice. Bxcimination of thu offoct of an aotivo F1t, F1t3 LID, in B ooII dofolopmsnt. 7Ra mice. To test whether ectopic EBF or active Stat5 can rescue B cell development in Flt3LlL- E RU) m coo 7Ra To test whether ectopic EBF or active Stat5 can rescue B cell development in FIt3L"'IL- !alta. !pita n:g, iA 8 9s11 aevelePFR8At iii a;? Since the time is limited, our goal in Aiim 2d isat leastttoelluciidattetthecandididattemolleculelestthatt are associated with the 414-441 region of IL-7RcacandarereininvolvlvedininprrottecttioionofSttatSt5from proteolysis upon activation ofsignal cascades via lIL-7R.